Superfrost plus slides

For histopathological investigation, the skull was removed and the head was fixed in 4% neutral‐buffered formalin for at least 1 week followed by decalcification for 3 days in Formical 2000 (Decal, Tallman, NY). From all heads, eight coronal head sections were obtained, embedded in paraffin wax and cut at 3 or 5 µm thick slices, respectively, for further histological and immunohistochemical evaluation [28 (link)]. The slices were mounted on Super‐Frost‐Plus‐Slides (Carl Roth GmbH, Karlsruhe, Germany) and stained with hematoxylin–eosin for detailed neuropathological analysis of CNS inflammation.

Retinas were digested using trypsin as previously described with slight modifications [27 ]. Briefly, after enucleation the eyecup was fixed in 4% PFA for 45 min, then the retina was isolated and further fixed in 4% PFA overnight. The next day, the retinas were washed (5x) with distilled water. After overnight shaking in water at room temperature, retinas were transferred into 24-well plates and incubated in 3% trypsin (#9002-07-7, Affymetrix USB, Ohio, USA) in 0.1 M Tris buffer (pH 7.8) at 37 °C for 90 min. The inner limiting membrane was removed with scissors and then the vasculature isolated by several washing steps. The retinal vasculature system was flat mounted on Thermo Scientific™ SuperFrost Plus™ slides and stained with H&E (H&E fast staining kit, #9194.1, Carl Roth). Microscopy was performed at the Core Facility Bioimaging of the Biomedical Center at the LMU with a Leica DM6 FS microscope. Bright field images were recorded with Leica DMC2900 CMOS camera with an image pixel size of 145 nm. A 20x/0.8 objective was used for quantification and overview image of the network and a 40x/0.95 objective for detailed view of the vascular network.